Mast cells play an important role at the early stages of immunological response to bacterial infections and parasite infestations. One of the major mast cell proinflammatory mediators is TNF‐α. Mast cells are considered the only cells capable of storing TNF‐α in cytoplasmic granules and rapidly releasing it upon activation. To determine what pathway is utilized to direct TNF‐α to cytoplasmic granules and what motifs are responsible for the sorting process, we constructed a fusion protein covering the full sequence of TNF‐α, N‐terminally fused to enhanced green fluorescent protein (EGFP). In rodent mast cells, such protein was sorted to secretory granules, and this process was inhibited by both brefeldin A and monensin. Considering the relationship between lysosomes and secretory granules and following TNF‐α sequence analysis, it was determined whether TNF‐α is sorted through the mannose‐6‐phosphate receptor (MPR)‐dependent pathway. We observed that ammonium chloride and tunicamycin blocked TNF‐α‐EGFP fusion protein delivery to secretory granules. In situ mutagenesis experiments confirmed the necessity of N‐linked glycosylation for efficient sorting of TNF‐α into rodent mast cell granules. In this work we established that TNF‐α travels from the ER to mast cell granules via a brefeldin A‐ and monensin‐sensitive route, utilizing the MPR‐dependent pathway, although this dependency does not seem to be absolute.

Chemical modification of known, effective drugs is one method to improve chemotherapy. Thus, the object of this study was to generate melphalan derivatives with improved cytotoxic activity in human cancer cells (RPMI8226, HL60 and THP1). Several melphalan derivatives were synthesised, modified in their two important functional groups. Nine analogues were tested, including melphalan compounds modified: only at the amino group, by replacing the amine with an amidine group containing a morpholine ring (MOR-MEL) or with an amidino group and dipropyl chain (DIPR-MEL); only at the carboxyl group to form methyl and ethyl esters of melphalan (EM-MEL, EE-MEL); and in a similar manner at both functional groups (EM-MOR-MEL, EE-MOR-MEL, EM-DIPR-MEL, EE-DIPR-MEL). Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (comet assay) and the ability to induce apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling, TUNEL, phosphatidylserine externalisation, chromatin condensation, activity of caspases 3/7, 8 and 9 and intracellular concentration of calcium ions) in comparison with the parent drug. Almost all derivatives, with the exception of MOR-MEL and DIPR-MEL, were found to be more toxic than melphalan in all cell lines evaluated. Treatment of cultures with the derivatives generated a significant higher level of DNA breaks compared to those treated with melphalan, especially after longer incubation times. In addition, all the melphalan derivatives demonstrated a high apoptosis-inducing ability in acute monocytic and promyelocytic leukemia cells. This study showed that the mechanism of action of the tested compounds differed depending on the cell line, and allowed the selection of the most active compounds for further, more detailed investigations.

Background: H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA). Methods: In the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis. Results: The H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively. Conclusions: The H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.